| Objective:Colorectal cancer is one of the most common malignancies,and its mortality and incidence rate are among the top three malignant tumors,which is a serious threat to human health.OXAbased chemotherapy is the first-line treatment for patients with progressive and metastatic colorectal cancer,however,chemoresistance is a serious problem in clinical practice,which seriously impacts the survival benefit of colorectal cancer patients.OXA chemoresistance is a very complex process,which is affected by many factors,but its specific molecular mechanism is still unclear.Therefore,revealing the molecular mechanism of OXA chemotherapy resistance in colorectal cancer and searching for new predictive biomarkers has always been a hotspot and an urgent problem in the treatment of colorectal cancer.It is also crucial to improve the treatment effect and prognosis of colorectal cancer.N6-methyladenosine(m6A)is a kind of dynamically reversible and post-translational modification of RNA prevalent in eukaryotes.m6A modification imbalance plays an important role in the development of colorectal cancer,but its role in OXA chemoresistance in colorectal cancer is unclear.In this study,we aim to screen and identify the key gene of m6A modification that regulates OXA chemoresistance in colorectal cancer from the perspective of mRNA epigenetics,to clarify the correlation between its expression and the response to OXA chemotherapy in colorectal cancer patients,to confirm its role in the proliferation of colorectal cancer cells and OXA resistance,and to investigate the regulatory mechanism of their onset of m6A methylation,so as to provide new thoughts for revealing new mechanisms of OXA resistance in colorectal cancer and overcoming drug resistance.Methods:1.Analysis of the expression differences between colorectal cancer OXA resistant cell line HCT11 6/OXA and its parental cells in terms of m6A modification and its related regulators.(1)HCT116/OXA,a colorectal cancer OXA resistant cell line,was established by the concentration gradient method,and the OXA resistance of the cells was detected by morphological observation and CCK8 assay.(2)The level of m6A modification in OXA resistant cells HCT116/OXA and their parental cells HCT116 were analyzed by Dot blot and m6A methylation quantitative assay.(3)The expression of m6A methylation-related regulators in OXA resistant cell line HCT116/OXA and its parental cells HCT116 were analyzed by qRT-PCR and Western blot.2.Screening and identification of the key m6A modified genes related to OXA resistance in colorectal cancer.(1)The differential m6A methylation modification map of colorectal cancer OXA resistant cell line HCT116/OXA and its parent cells HCT116 was established by MeRIP-seq,and the potential regulatory pathway of the differential m6A modification genes were predicted by bioinformatics analysis.(2)The transcription levels of HCT116/OXA cells and HCT116 cells were analyzed by RNA-seq,and the candidate m6A modification genes whose m6A modification levels were simultaneously up-regulated with transcription levels in HCT116/OXA cells were screened by MeRIP-seq and RNA-seq overlap analysis.(3)The correlation between the expression of the above candidate m6A modified genes and the prognosis of colorectal cancer was verified through the UALCAN online analysis database,and three candidate m6A modified genes related to the poor prognosis of colorectal cancer patients were obtained.(4)The expression of three candidate genes in OXA resistant cells was further verified by qRT-PCR,and the most significantly up-regulated ZDHHC11,a palmitoyl transferase,in HCT116/OXA cells was selected for subsequent studies;Western blot and MeRIP-qPCR were used to verify the expression of ZDHHC11 and the level of m6A methylation in OXA resistant cells.3.The analysis of the correlation between the expression of palmitoyl transferase ZDHHC11 and chemotherapy response in colorectal cancer patientsThe RNA expression profile data of tumor tissues from colorectal cancer patients with FOLFOX chemotherapy protocol response information was downloaded from the GEO database GSE28702,and the expression of ZDHHC11 in non-response and response colorectal cancer tissues was analyzed,as well as the correlation with FOLFOX chemotherapy resistance.The potential of ZDHHC11 as a predictive marker of FOLFOX chemotherapy resistance was evaluated by receiver operator characteristic(ROC).4.Effects of palmitoyl transferase ZDHHC11 on proliferation and OXA resistance of colorectal cancer cells.(1)We constructed ZDHHC11 stable overexpression and knockdown cell lines.The overexpression and knockdown efficiency were verified by qRT-PCR and Western blot,and the biological functions of ZDHHC11 in colorectal cancer cell proliferation and OXA resistance were investigated by CCK-8 assays and colony formation assays.(2)The differential genes after knockdown of ZDHHC11 in HCT116 cells obtained from RNA-seq were analyzed through GO and KEGG enrichment.It is speculated that ZDHHC11 may be involved in the signal pathway of colorectal cancer progression and drug resistance.5.Mechanism of m6A modification regulating the expression of ZDHHC11(1)The GEPIA database was used to analyze the correlation between ZDHHC11 and METTL3 as well as IGF2BP1/2/3 in colorectal cancer tissues.The expression of METTL3 in non-response and response colorectal cancer tissues in GSE28702 database,and the correlation with FOLFOX chemotherapy resistance were analyzed.(2)METTL3 was overexpressed or knocked down in HCT116 and HCT116/OXA cells by adenovirus infection and small interference transfection,respectively.The effect of METTL3 on the expression of ZDHHC11 and the level of its mRNA m6A was verified by qRT-PCR,Western blot and MeRIP-qPCR.(3)The effect of overexpression or knockdown of METTL3 in HCT116 and HCT116/OXA cells on the stability of ZDHHC11 mRNA was detected by the actinomycin D RNA stability assay.(4)IGF2BP3 was knocked down by small interference in HCT116/OXA cells.The actinomycin D RNA stability assay,qRT-PCR and Western blot were used to detect the regulatory effect of IGF2BP3 on the stability and expression of ZDHHC11 mRNA.Results:1.The IC50 of colorectal cancer OXA resistant cell line HCT116/OXA was significantly higher than that of its parental cell line HCT116 to OXA,and morphological changes were also observed,which can confirm that the OXA resistant cell line HCT116/OXA was successfully constructed.The results of Dot blot and m6A methylation quantitative assay showed that the level of RNA m6A modification was significantly higher in colorectal cancer OXA resistant cell line HCT116/OXA compared with HCT116,and was accompanied by an abnormal increase of m6A methyltransferase METTL3.2.The results of MeRIP-seq showed that there were significant differences in m6A methylation modification map between OXA resistant cells and its parental cells.We obtained 282 m6A peaks and 89 m6A modified genes that were specifically upregulated in OXA resistant cells.KEGG analysis showed that the genes upregulated in m6A modification levels in OXA resistant cells were mainly enriched in MAPK,P53 and other classical drug resistance signal pathways.Combined with RNA-seq analysis and survival analysis in TCGA database,three candidate genes were selected to meet the requirements of upregulated m6A modification levels and transcription levels in OXA resistant cells and closely related to poor prognosis of patients.Among them,palmitoyl transferase ZDHHC11 mRNA was upregulated most significantly in HCT116/OXA cells,and its m6A modification level and protein level were also significantly upregulated.3.GEO database analysis found that ZDHHC11 was relatively highly expressed in colorectal cancer tumor tissues of patients who did not respond to FOLFOX chemotherapy before treatment,and was associated with poor response to chemotherapy.ROC curve analysis showed that ZDHHC11 had the potential to identify patients with OXA resistance tendency.4.In vitro function experiments showed that overexpression of ZDHHC11 could promote the proliferation,colony formation and OXA resistance of HCT116 cells,whereas knockdown of ZDHHC11 could significantly inhibit the proliferation,colony formation and OXA resistance of HCT116/OXA cells in vitro.GO and KEGG enrichment analysis of the differential genes of ZDHHC11 knockdown in HCT116 cells obtained from RNA-seq showed that the differential gene was related to cell proliferation,cell adhesion,stress regulation and other functions,and was significantly enriched in the signal pathways closely related to tumor progression and drug resistance such as MAPK,p53,Wnt,platinum chemotherapy resistance,etc.5.TCGA database analysis showed that the expression of METTL3 and ZDHHC11 was positively correlated in colorectal cancer tissues,and GEO database analysis found that their expression was abnormally high in chemoresistant tissues containing OXA,which was closely related to poor FOLFOX chemotherapy response.Overexpression of METTL3 promoted the m6A methylation level of ZDHHC11 mRNA,enhanced its mRNA stability,and upregulated its mRNA and protein expression levels.Conversely,knockdown of METTL3 could inhibit the m6A methylation level of ZDHHC11 mRNA,weaken its mRNA stability,and downregulated its mRNA and protein expression levels.6.TCGA database analysis showed that there was a positive correlation between the expression of IGF2BP3 and ZDHHC11 in colorectal cancer tissues,and knockdown of IGF2BP3 could inhibit the mRNA and protein expression levels of ZDHHC11,indicating that METTL3 might maintain the stability of ZDHHC11 through IGF2BP3.Conclusions:1.The m6A modification level and expression of ZDHHC11 mRNA were significantly upregulated in colorectal cancer OXA resistant cells,and relatively high expression in patients with FOLFOX chemoresistance,which was closely related to poor prognosis of colorectal cancer patients and poor response to FOLFOX chemotherapy,and had the potential to identify patients with FOLFOX chemoresistance.It is suggested that ZDHHC11 may be the key gene of m6A modification regulating OXA resistance in colorectal cancer.2.ZDHHC11 promotes the proliferation,colony formation and OXA resistance of colorectal cancer cells,and may play a role by regulating the signal pathway closely related to tumor progression and drug resistance,which may provide a basis for finding new intervention targets to reverse OXA resistance of colorectal cancer.3.METTL3 can upregulate the m6A modification level of ZDHHC11 mRNA,and may enhance its mRNA stability and expression through methylation reading protein IGF2BP3,indicating that METTL3-mediated m6A modification plays an important role in maintaining the high expression of ZDHHC11 in colorectal cancer cells. |
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